Fine mapping and marker development for the wheat leaf rust resistance gene Lr32

Abstract Wheat leaf rust is caused by the fungal pathogen Puccinia triticina and is one of the wheat diseases of concern globally. Among the known leaf rust resistance genes (Lr) genes, Lr32 is a broadly effective gene derived from the diploid species Aegilops tauschii coss. accession RL5497-1 and has been genetically mapped to chromosome arm 3DS. However, Lr32 resistance has not been utilized in current cultivars in part due to the lack of modern, predictive DNA markers. The goals of this study were to fine map the Lr32 region and develop SNP-based kompetitive allele-specific polymerase chain reaction markers. The genomic analysis was conducted by using doubled haploid and F2-derived mapping populations. For marker development, a 90K wheat chip array, 35K and 820K Axiom R SNPs, A. tauschii pseudomolecules v4.0 and International Wheat Genome Sequencing Consortium ReqSeq v2.1 reference genomes were used. Total 28 kompetitive allele-specific polymerase chain reaction and 2 simple sequence repeat markers were developed. The Lr32 region was fine mapped between kompetitive allele-specific polymerase chain reaction markers Kwh142 and Kwh355 that flanked 34–35 Mb of the diploid and hexaploid reference genomes. Leaf rust resistance mapped as a Mendelian trait that cosegregated with 20 markers, recombination restriction limited the further resolution of the Lr32 region. A total of 10–11 candidate genes associated with disease resistance were identified between the flanking regions on both reference genomes, with the majority belonging to the nucleotide-binding domain and leucine-rich repeat gene family. The validation analysis selected 2 kompetitive allele-specific polymerase chain reaction markers, Kwh147 and Kwh722, for marker-assisted selection. The presence of Lr32 along with other Lr genes such as Lr67 and Lr34 would increase the resistance in future wheat breeding lines and have a high impact on controlling wheat leaf rust.

Plant disease resistance genes have been classified into racespecific and nonrace-specific groups. The race-specific resistance follows the gene-for-gene model described by Flor (1955) that states that for each resistance (R) gene in the plant there will be a corresponding avirulence (AVR) gene in the pathogen. Some of the nonrace-specific resistance type genes are effective against many pathogens and were reported to be associated with a variety of genes encoding receptor kinases, transcription factors, membrane proteins, Kinase-START, and ATP-binding cassette (ABC) transporters (Singh et al. 2015). Many of the race-specific R genes in wheat were associated with the nucleotide-binding domain and leucine-rich repeat (NLR) gene family. To date, only 7 Lr genes have been cloned Lr1 (Cloutier et al. 2007), Lr10 (Feuillet et al. 2003), Lr21 (Huang et al. 2003), Lr22a (Thind et al. 2017), Lr34/ Yr18/Sr57/Pm38 (Krattinger et al. 2009), Lr67/Yr46/Sr55/Pm46 (Moore et al. 2015), and Lr14a (Kolodziej et al. 2021). Out of which, race-specific leaf rust genes Lr1, Lr10, and Lr21 belong to the NLR gene family. Lr14a was sequenced and was found to belong to ankyrin (ANK) transmembrane-like gene family, which is a first in wheat (Kolodziej et al. 2021). Genes conditioning race-specific resistance tend to lose their effectiveness as the pathogen population evolves virulence. For example, the P. triticina population in Canada has evolved virulence to Lr1, Lr2a, Lr10, Lr12, Lr13, Lr14a, and Lr21 which were all used in Canadian wheat cultivars to control leaf rust (McCallum et al. 2016). In contrast durable, race nonspecific, slow-rusting leaf rust resistance genes Lr34, Lr46, and Lr67 have proved to be long-lasting and are able to confer resistance against multiple pathogens and races (Singh et al. 1998;Krattinger et al. 2009;Moore et al. 2015;McCallum et al. 2016). High levels of durable leaf rust resistance have been achieved by combining race-specific and race-nonspecific resistance genes in the same cultivar, such as the Canadian cultivar Carberry with Lr2a, Lr16, Lr23, Lr34, and Lr46 (Bokore et al. 2022). Consequently, new gene combinations need to be introduced in the wheat cultivars to achieve the durable resistance and decrease the selection pressure on Pt populations for virulence against R genes. For combining multiple R genes, the availability of gene-based and functional molecular makers is ideal. Within the last few years, high-throughput genetic analysis has become more advanced with the development of single nucleotide polymorphism (SNP) chips (Wang et al. 2014;Allen et al. 2017) and the availability of reference genome resources (Luo et al. 2017;Maccaferri et al. 2019;Zhu et al. 2019) which has led to better markers for a higher number of genes and resources needed for gene cloning experiments.
Among the broadly effective leaf rust resistance genes (Lr21, Lr22a, Lr32, Lr52, and Lr60) identified at the Morden Research and Development Centre, AAFC, Morden (and the former Cereal Research Centre, Winnipeg): Lr32, Lr52, and Lr60 have not been deployed in Canadian commercial cultivars (Thomas et al. 2010) and Lr22a has been deployed in 3 cultivars (AC Minto, 5500HR, and 5600HR) that occupied minimal acreage (Hiebert et al. 2007). The seedling leaf rust resistance gene Lr32 was discovered in A. tauschii coss. (accession number RL5497-1) and was mapped using simple sequence repeat (SSR) markers on chromosome arm 3DS. It was physically located in deletion bin 6 (3DS6-0.55-1.00) which spans $75 cM of genetic distance (Kerber 1987(Kerber , 1988Sourdille et al. 2004;Thomas et al. 2010). The SSR map was not dense, and while some SSRs were very closely linked to Lr32, markers that reliably flanked the gene had large genetic distances to Lr32. Hence, the objectives of this study were: (1) to develop the new kompetitive allele-specific polymerase chain reaction (PCR) (KASP) markers for Lr32 suitable for marker-assisted selection (MAS) and (2) fine map the Lr32 region.

Plant material
Wheat line BW196 (¼Katepwa*6//RL5713/2*MarquisK) was found to be heterogeneous for Lr32 based on phenotype and molecular marker data (Thomas et al. 2010). Therefore, for the development of mapping populations, a single resistant reselection (BW196R) was used as a resistance parent as explained in Thomas et al. (2010). A double haploid (DH) population (n ¼ 244), an initial F 2 (n ¼ 196) population, and an expanded large F 2 (n % 2,000) population were developed from a cross between the susceptible parent Thatcher and the resistant line BW196R. The initial, smaller F 2 population and the expanded F 2 population were generated from the same cross, but there was no overlap in individuals between the 2 populations. The F 3 families were developed from the large F 2 population lines based on markers flanking the Lr32 region (recombinants were selected, detailed description in the Genotyping section). From these recombinant F 3 progenies, fixed recombinant lines were selected based on the marker genotypes. The DH population was developed using the maize pollination method as described by Thomas et al. (1997). For the validation of the newly developed MAS markers, a panel of 32 wheat lines was used which consisted of material ranging from F 1 progeny to advanced generations (with Lr32 plus additional Lr genes), Lr32-lacking susceptible check Neepawa, and an Lr32 carrier (BW196R).

Leaf rust testing
The leaf rust phenotyping of the parents, Thatcher and BW196R, DH population, F 2 population, F 3 progenies from the initial F 2 population, and fixed recombinants derived from expanded F 2 population was done with the urediospores of Pt race 12-3 MBDS at the seedling stage following the method described by McCallum and Seto-Goh (2003). Briefly, the plants were inoculated at the first leaf stage ($7 day old) with urediospores of Lr32 avirulent Pt race 12-3 MBDS suspended in light mineral oil (McCallum et al. 2021). After inoculation plants were kept in humidity-maintained chambers for 16 h at 18-20 C and subsequently transferred to the greenhouse which was kept at 20 6 2 C. After 12-14 days plants were scored in the scale of 0-4 (0, 1, 2, 3, 4) where 0-2 were considered resistant and 3-4 considered susceptible, the symbols "þ" and "À" were used to note pustule sizes that were larger or smaller than typically observed for a given infection type (McCallum et al. 2021).

Genotyping and marker development
The DNA extraction of parents and mapping populations (DH, F 2 , and fixed recombinants) was done using a modified ammonium acetate method (Pallota et al. 2003). The DH population was genotyped using 90K iSelect SNP array (Wang et al. 2014) and 8 SSR markers, barc128, barc135, barc376, cfd34, gwm2, gwm183, wmc43, and wmc539 reported in the Thomas et al. (2010). A linkage map was constructed using the software MapDisto 1.8.2 (Lorieux 2012) and genetic distances were calculated using the Kosambi mapping function (Kosambi 1943). After locating the Lr32 region on the linkage map, the associated SNPs were converted into KASP markers (Tables 1 and 2). Total 17 KASP were developed from the 90K SNP array and genotyping was done according to the procedure described in Kassa et al. (2016). One SSR marker Swh28 was developed from the A. tauschii pseudomolecules v4.0 genebased region by using the software package Batchprimer3 (You et al. 2008) (Table 3). In addition, FASTA sequences of NBS-LRR gene regions from International Wheat Genome Sequencing Consortium (IWGSC) ReqSeq v1.0 were also used to develop the SSR markers, and out of which Swh45 is polymorphic between parents (Table 3). The F 2 population (n ¼ 196) was genotyped with the 12 KASP developed in the DH population and 3 SSR markers barc135, Swh28, and Swh45. After that, codominant flanking DNA markers were selected and these markers were used for the screening of $2,000 F 2 individuals to identify plants with a recombination event within the interval carrying Lr32. F 3 progeny (minimum 16 progenies/family) from each recombinant F 2 plant was tested with the flanking markers to select progeny fixed for recombination events and were genotyped with the remaining KASP markers.
To increase the resolution of the Lr32 spanning genetic region, an additional 11 KASP markers were developed or selected from the CerealsDB database, 35K and 820K Axiom R SNPs, and A. tauschii (accession AL8/78) pseudomolecules v4.0 (Tables 1 and 2). First, the flanking KASP markers source sequences were aligned against the IWGSC ReqSeq v1.0 via using the basic local alignment search tool (BLAST) (Altschul et al. 1997) and the Jbrowse portal was used to explore the region. Within that region, 2 SNPs (BS00070468 and BS00072718) were selected and associated KASP markers Kwh489 and Kwh491 (Tables 1 and 2) were used for genotyping the fixed recombinant lines (CerealsDB database; Wilkinson et al. 2012). Within that region, additional 35K and 820K Axiom R SNPs were also selected to develop KASP markers (Allen et al. 2017). Total 6 KASP markers, Kwh605, Kwh617, Table 1. Lr32 region-associated kompetitive allele-specific (KASP) PCR markers source, SNP name, primer sequence information.
Marker name SNP name/gene ID Primer name Tail-1 (FAM tail-GAAGGTGACCAAGTTCATGCT), Tail-2 (VIC tail-GAAGGTCGGAGTCAACGGATT)  Kwh628, Kwh721. Kwh722, and Kwh727, were developed from the Axiom SNPs and used to genotype the fixed recombinants (Tables 1 and 2). Moreover, the flanking KASP markers source sequences were also used to select the A. tauschii (accession AL8/ 78) pseudomolecules v4.0 (Luo et al. 2017) gene regions. Out of which, LRR motif-containing gene regions were selected and BLASTed against the IWGSC RefSeq v1.0 assembly. After aligning the FASTA sequence from both assemblies, SNPs were identified and 3 KASP markers, Kwh645, Kwh646, and Kwh662, were manually developed by using the Primer3 software package (Ye et al. 2012). The source sequences of KASP and SSR markers were used to determine the physical coordinates on the A. tauschii pseudomolecules v4.0 and IWGSC ReqSeq v2.1. The mode of inheritance (dominant/codominant) of the markers developed in the current study was determined on the F 2 population. To validate these markers a genetic analysis was conducted with F 1 and prebreeding germplasm carrying Lr32 (Supplementary Table 1). The Aet_v4.0 and IWGSC ReqSeq v2.1 reference genomes data were used to extract the disease resistance candidate genes between flanking markers Kwh142 and Kwh355.

Results
The leaf rust screening showed that the susceptible parent Thatcher had IT 3 and the resistant parental line BW196R had IT 1À (Fig. 1). The DH population phenotypic ratio fitted that expected for a single gene (109R:134S; v 2 ¼ 2.25, P ¼ 0.11). The initial F 2:3 population had 51 resistant (HR), 96 segregating (hetero), and 49 susceptible (HS) families which also fitted a single gene ratio (v 2 ¼ 0.12, P ¼ 0.94). The DH and F 2:3 population IT scores ranged between 1À and 3 (HR ¼ 1À/12/12, hetero ¼ 2À to 3À, HS ¼ 3/3þ) ( Supplementary Files 1 and 2). Linkage mapping in the DH population using 90K SNP markers and SSR markers developed for the Lr32 region resulted in a genetic map of chromosome arm 3DS that spanned 37.4 cM and consisted of 39 SNP and 8 SSR markers ( Fig. 2 and Supplementary Files 1 and 2). Lr32 was mapped at position 17.8 cM and cosegregated with SSRs wmc43 and barc135. In addition, a total of 24 SNP markers cosegregated with Lr32 ( Supplementary Files 1 and 2). This region was flanked by SNPs IWB32645 and IWB18374 (Fig. 2). Genetic analysis conducted on the F 2 population with 12 KASP and 3 SSR markers developed a chromosome arm 3DS linkage map of 6.73 cM (Fig. 2). The KASP markers Kwh142 and Kwh355 positioned at 14.5 and 22.1 cM on the chromosome 3DS DH linkage map were selected as the flanking markers and used for screening the $2,000 F 2 progenies (Fig. 2). Selected F 2 progeny were used to generate F 3 progeny. Based on genotypic and phenotypic screening of these F 3 progenies, 106 fixed recombinants (i.e. both homologs carried the same recombination event between the flanking markers with no heterozygous alleles for the flanking markers) were identified (Fig. 3). Further genetic analysis was done on these 106 fixed recombinants with the remaining 26 KASP and 2 SSR markers were developed from 90K SNP chip, Axiom SNPs, A. tauschii gene-based regions and CerealsDB database ( Fig. 3 and Tables 1 and 2). The overall genomic analysis conducted on the fixed recombinants fine-mapped Lr32 to a region spanning 2.57 cM between the flanking markers. In the fine mapping population, a total of 18 KASP and 2 SSR markers cosegregated with Lr32 and were flanked by KASP markers Kwh 722 and Kwh 638 (Supplementary File 3). Mapping of KASP markers source sequences on reference genomes showed that the Lr32 region is collinear in both diploid and hexaploid genome assemblies. Total 11 and 10 candidate genes belonging to the gene's classes typical of disease resistance genes were identified between the flanking markers Kwh142 and Kwh355 in the IWGSC RefSeq v2.1 and A. tauschii pseudomolecules v4.0. reference genomes, respectively (Table 4).

Discussion
The phenotyping results showed that the DH and initial F 2 populations segregated for 1 gene. The collinearity of SSR and KASP markers in the DH and F 2 mapping populations and their corresponding physical order on reference genomes demonstrate the marker order was robust (Figs. 1 and 3). In addition, large numbers of 90K SNP/KASP markers cosegregating with the Lr32 resistance phenotype in the 3 populations (DH, F 2 , and fixed recombinants) analyzed in the current study indicates that Lr32 region is associated with a linkage block with recombination restriction. Physical mapping of the SNP source sequences on the A. tauschii pseudomolecules v4.0 and IWGSC ReqSeq v2.1 reference genomes showed that the Lr32 region spanned about 34-36 Mb between the flanking markers Kwh142 and Kwh355, which clearly showed that the physical size of these segments is large (11-13 Mb/cM) (Fig. 3). The cosegregating markers with Lr32 phenotype represent 26-28 Mb on both reference genomes (Fig. 3). These results explained the limitation to further increase the resolution of the Lr32 region. In addition, it also indicates a lower recombination frequency associated with alien R genes. Among the candidate genes identified from the reference genomes, NLR is the most abundant one. Besides that Protein Kinase and RGA types genes were also identified. Both classes of NLR genes, Tollinterleukin receptor type (TIR) and N-terminal coiled-coil (CC) were identified. Whereas, to date only CC-NBS-LRR has been identified in wheat for disease resistance (reviewed in Md. Hatta et al. 2019).
The validation analysis done with the F 1 and advanced prebreeding lines showed that the 2 KASP markers Kwh147 and Kwh722 are codominant and able to differentiate between plants carrying Lr32 in the heterozygous/homozygous states (Fig. 4). Marker Kwh340 also detects the presence of the Lr32-resistant allele; however, clusters are not well differentiated. These markers should be useful for the selection of Lr32 in wheat breeding programs. Pyramiding Lr32 with other effective genes such as adultplant resistance genes Lr34, Lr46 or Lr67 should help to delay the evolution of Lr32 virulent P. triticina isolates. To date virulence to Lr32 has not been detected in Canada (McCallum et al. 2021), although there is a report for virulence in South Africa (Pretorius Fig. 2. a) The chromosome 3DS deletion Bin6. b) The SSR markers based chromosome arm 3DS linkage map spanning Lr32 developed in the DH population (Thomas et al. 2010). c) The Lr32 associated chromosome arm 3DS 90K SNP's linkage map developed in the Thatcher Â BW196R DH population. d) The 3DS linkage map developed in the Thatcher Â BW196R F 2 population (n ¼ 196) by using 12 KASP and 3 SSR markers. Common SSR markers between 3DS map developed by Thomas et al. (2010) and current study were shown in Blue fonts and KASP markers developed from the 90K SNPs were represented with the green fonts. Fig. 3. Collinearity of Lr32 high resolution genetic region-associated KASP markers (b) on the Aegilops tauschii (Aet v 5.0) (a) and 3DS Chinese spring reference (Refseq v2.1) genomes. The high-resolution mapping was done by using 106 fixed recombinants. and Bender 2010). The absence of virulence to Lr32 in Canada may be due to the fact that this gene has not been deployed in a commercial wheat cultivar to drive evolution of virulence in the pathogen population. The resistance gene Lr67 was recently shown to have a significant interaction with Lr32 when the 2 genes were in combination in a population that segregated for these 2 genes in the Thatcher background (McCallum and Hiebert 2022). The interaction between Lr34 and Lr32 was not significant in a population that segregated for both these genes; however, lines with both genes were more resistant than lines with either-gene alone. Using the molecular markers developed in this study Lr32 can be successfully incorporated into wheat lines, such as Carberry with Lr34, Lr46, and other resistance genes, that already have a good base of genetic resistance (Bokore et al. 2022). Deploying Lr32 in isolation would likely lead to the evolution of virulence on Lr32 as was seen for Lr21, a similar resistance gene also derived from A. tauschii, both in the USA (Kolmer and Anderson 2011) and Canada (McCallum et al. 2017).
In conclusion, the development of these new functional markers will accelerate integration of Lr32 into the breeding lines and will be helpful in MAS to develop the future wheat cultivars with durable resistance.

Data availability
Seed is available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Genotypic and phenotypic data available in supplementary files.
Supplemental material is available at G3 online.